[Construction, expression and purification of UreB-Omp11 fusion protein of Helicobacter pylori and its immunocompetence]

Abstract

Aim: To construct H.pylori vaccine candidate strain expressing UreB-Omp11 recombinant fusion protein of H.pylori. To express and purify the fusion protein UreB-Omp11 and to determine its immunocompetence.

Methods: The two genes were amplified by PCR, and the fusion gene ureB-omp11 was amplified by over lap extension PCR and then cloned into the fusion expression vector pET30a(+), pET28a(+) and pMAL-c2X. The appropriate expression system was selected, and the recombinant UreB-Omp11 fusion protein was expressed and indentfied by SDS-PAGE and Western blot analysis. Then the fusion protein was purified by MBP affinity chromatography and the purity was indentfied by SDS-PAGE. Then the fusion protein was immunized to mice. The immunized mice sera were analyzed by Western blot with purified fusion protein.

Results: The ureB-omp11 fusion gene was correctly insected into pET30a(+) and confirmed by Enzyme digestion and sequencing analysis; Results in SDS-PAGE and optical density scanning demonstrated that this fusion protein MBP-UreB-Omp11 was expressed in the recombinant strain of E.coli TB1(pMAL-ureB-omp11). The fusion protein UreB-Omp11 was recognized by the mice sera immunized by H.pylori, the human sera infected with H.pylori and The purity of fusion protein was 90% after purification. The fusion protein purified could be recognized by corresponding antibody of mice sera immunized by this fusion piotein, This fusion protein has strong immunoantigenicity and immunoreactivity.

Conclusion: The prokaryotic expression system TB1 (pMAL-c2X-ureB-omp11) was successfully constructed and selected. The results obtained lay the foundation for research on development of protein and DNA vaccine of Hp.